Summary Protein phosphatase PP2C encompasses a family of a dozen human enzymes with related sequences that fold into a unique 3D structure that exhibits metal ion dependence, a catalytic mechanism and substrate specificity distinct from other types of protein Ser/Thr phosphatases. The PP2C are fundamentally important, with multiple genes conserved from yeast to human, and gene knockout phenotypes implicate PP2C in control of responses to radiation damage and other forms of cellular stress. Transcription of the PP2C isoform known as Wip1 (Wild type p53 Induced Phosphatase; or PP2C or PPM1D) is controlled by the tumor suppressor p53. Deletion of this gene results in activation of the DNA damage response pathway and suppresses tumor formation by oncogenes. To date there are no potent synthetic or natural product small molecule inhibitors or activators for any PP2C, which has hampered investigations of their biological functions and prevented development of drugs that target this family of regulatory enzymes. This project proposes a triple threat approach, developing in parallel HTS-compatible assays for three different members of the PP2C family. The plan is to produce recombinant, purified phosphatases PP2C, PP2C and PP2C (Wip1) and establish optimum conditions for HTS assay using chromogenic and phosphopeptide substrates. This approach builds in secondary screening for selectivity and isoform specificity and has the potential to identify both broad spectrum and highly specific chemical modulators of PP2C activity. A second Aim is to develop and optimize a new In-Cell Western HTS assay that quantitates simultaneous staining with two different antibodies by dual wavelength infrared detection. This live cell assay will detect cell-permeable compounds that affect PP2C activity against endogenous substrates. The availability of small molecule modifiers would be a major advance that would benefit investigations of biological functions of different PP2C and pave the way for development of novel compounds with medicinal properties. Project Narrative Human physiology is governed by internal hormone signals and changes in the external environment. Cells respond to these signals through networks of protein kinase and phosphatase enzymes that dynamically switch on/off various processes by the addition and removal of phosphate groups on thousands of different proteins. Human diseases arise from imbalances between these kinases and phosphatases and new targeted therapies in clinical use are chemicals that block the activity of these enzymes. A family of phosphatase enzymes called PP2C regulates responses to DNA damage and cell stress. This project seeks to produce specific and dependable assays for PP2C to screen collections of thousands of chemicals and identify novel activators or inhibitors. Chemical modifiers of PP2C have potential medicinal applications and will be used as research tools to study signaling in normal and cancer cells. [unreadable] [unreadable] [unreadable]